# dic4 pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Dic4 Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

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Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3"

**Article Title: **Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

**Journal: **International Journal of Molecular Sciences

**doi: **10.3390/ijms24021125

**Figure Legend Snippet:**Phospholipid-binding abilities of Arap3-PH1 domain analyzed using liposome pull-down assay and SPR measurements. ( A ) Schematic representation of human Arap3 protein. The first PH domain of Arap3 is marked in cyan. ( B ) Sequence alignment of Arap3-PH1 orthologs in vertebrates and human Arap1-PH1, Arap2-PH1. Sequence accession number in the Uniprot database are: human, Q8WWN8; bovine, E1BBA0; mouse, Q8R5G7; zebrafish, A0A140LH27; human Arap1, Q96P48; human Arap2, Q8WZ64. Alignment was performed using Clustal X and illustrated with ESPript 3.0. Strictly conserved (white letters filled with red color) and conservatively substituted (red letters with blue box) residues are denoted. The secondary structure element for human Arap3-PH1 is labeled on the top. The KXnQXR motif are marked by black dots. ( C ) Arap3-PH1 (20 μg) mixed with liposomes (640 μg) composed of 98% PC as the fixed component and 2% of specific phospholipids, respectively. Proteins in the absence of liposome were used as a control. After centrifugation, the pellet (P) and supernatant (S) were analyzed by SDS/PAGE and Coomassie. ( D ) SPR measurements of the binding affinities of the Arap3-PH1 domain for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2. The upper panel shows representative sensorgrams of diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) when mixed with Arap3-PH1. Data were collected by injecting increasing concentrations of diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 samples over Arap3-PH1 proteins immobilized on the surface of a CM5 biochip. The lower panel shows representative binding curves fitting for diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) during their interaction with Arap3-PH1. A one-site binding model was utilized to fit the curves. The experiment was carried out in triplicate. The KD value is presented as mean ± SD, n = 3.

**Techniques Used: **Binding Assay, Pull Down Assay, Sequencing, Labeling, Centrifugation, SDS Page

**Figure Legend Snippet:**Crystallographic data collection and refinement statistics.

**Techniques Used: **

**Figure Legend Snippet:**Structure of the Arap3-PH1 domain in complex with diC4-PI(3,4,5)P3. ( A ) Cartoon diagram of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Arap3-PH1 is colored turquoise, with secondary structures labeled. The loops of β1/β2 and β6/β7 that interact directly with diC4-PI(3,4,5)P3 are colored blue. The diC4-PI(3,4,5)P3 (gold) is shown in stick mode. ( B ) Surface electrostatic potential of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Blue areas, positive; red areas, negative. ( C ) Detailed interactions of the diC4-PI(3,4,5)P3 with Arap3-PH1 domain. The side chains of crucial residues are shown in stick mode and labeled, respectively. The phosphate groups on diC4-PI(3,4,5)P3 are also labeled. Selected hydrogen bonds or salt bridges are shown as dotted lines.

**Techniques Used: **Labeling

**Figure Legend Snippet:**The binding interfaces of Arap3-PH1 for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 revealed by NMR titration. ( A ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in the increasing amounts of diC4-PI(3,4,5)P3. The molar ratios of the protein to diC4-PI(3,4,5)P3 are shown in the inset: 1:0 (black), 1:0.25 (turquoise), 1:0.5 (lime green), 1:0.75 (orange), 1:1 (pink) and 1:1.25 (red). ( B ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in increasing amounts of diC4-PI(4,5)P2. The molar ratios of the protein to diC4-PI(4,5)P2 are shown in the inset: 1:0 (black), 1:0.5 (royal blue), 1:1 (turquoise), 1:2 (lime green), 1:4 (orange), 1:6 (pink) and 1:8 (red). ( C ) The chemical shift perturbations (CSPs) of each residue during NMR titrations (up, diC4-PI(3,4,5)P3 titration; down, diC4-PI(4,5)P2 titration) are calculated and shown with the secondary elements on top. White dots indicate pro residues. Black dots indicate residues with no data. The mean value and the mean value plus one standard deviation are indicated by dash and solid lines, respectively. Residues with CSPs between mean value and mean value plus one standard deviation are colored gold, and above mean value plus one standard deviations are colored red. ( D , E ) Surface representations of the Arap3-PH1 structure with the perturbed residues upon binding to diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 are colored and labeled.

**Techniques Used: **Binding Assay, Titration, Standard Deviation, Labeling

# dic4 pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Dic4 Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/dic4 pi 4 5 p2/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3"

**Article Title: **Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

**Journal: **International Journal of Molecular Sciences

**doi: **10.3390/ijms24021125

**Figure Legend Snippet:**Phospholipid-binding abilities of Arap3-PH1 domain analyzed using liposome pull-down assay and SPR measurements. ( A ) Schematic representation of human Arap3 protein. The first PH domain of Arap3 is marked in cyan. ( B ) Sequence alignment of Arap3-PH1 orthologs in vertebrates and human Arap1-PH1, Arap2-PH1. Sequence accession number in the Uniprot database are: human, Q8WWN8; bovine, E1BBA0; mouse, Q8R5G7; zebrafish, A0A140LH27; human Arap1, Q96P48; human Arap2, Q8WZ64. Alignment was performed using Clustal X and illustrated with ESPript 3.0. Strictly conserved (white letters filled with red color) and conservatively substituted (red letters with blue box) residues are denoted. The secondary structure element for human Arap3-PH1 is labeled on the top. The KXnQXR motif are marked by black dots. ( C ) Arap3-PH1 (20 μg) mixed with liposomes (640 μg) composed of 98% PC as the fixed component and 2% of specific phospholipids, respectively. Proteins in the absence of liposome were used as a control. After centrifugation, the pellet (P) and supernatant (S) were analyzed by SDS/PAGE and Coomassie. ( D ) SPR measurements of the binding affinities of the Arap3-PH1 domain for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2. The upper panel shows representative sensorgrams of diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) when mixed with Arap3-PH1. Data were collected by injecting increasing concentrations of diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 samples over Arap3-PH1 proteins immobilized on the surface of a CM5 biochip. The lower panel shows representative binding curves fitting for diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) during their interaction with Arap3-PH1. A one-site binding model was utilized to fit the curves. The experiment was carried out in triplicate. The KD value is presented as mean ± SD, n = 3.

**Techniques Used: **Binding Assay, Pull Down Assay, Sequencing, Labeling, Centrifugation, SDS Page

**Figure Legend Snippet:**Crystallographic data collection and refinement statistics.

**Techniques Used: **

**Figure Legend Snippet:**Structure of the Arap3-PH1 domain in complex with diC4-PI(3,4,5)P3. ( A ) Cartoon diagram of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Arap3-PH1 is colored turquoise, with secondary structures labeled. The loops of β1/β2 and β6/β7 that interact directly with diC4-PI(3,4,5)P3 are colored blue. The diC4-PI(3,4,5)P3 (gold) is shown in stick mode. ( B ) Surface electrostatic potential of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Blue areas, positive; red areas, negative. ( C ) Detailed interactions of the diC4-PI(3,4,5)P3 with Arap3-PH1 domain. The side chains of crucial residues are shown in stick mode and labeled, respectively. The phosphate groups on diC4-PI(3,4,5)P3 are also labeled. Selected hydrogen bonds or salt bridges are shown as dotted lines.

**Techniques Used: **Labeling

**Figure Legend Snippet:**The binding interfaces of Arap3-PH1 for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 revealed by NMR titration. ( A ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in the increasing amounts of diC4-PI(3,4,5)P3. The molar ratios of the protein to diC4-PI(3,4,5)P3 are shown in the inset: 1:0 (black), 1:0.25 (turquoise), 1:0.5 (lime green), 1:0.75 (orange), 1:1 (pink) and 1:1.25 (red). ( B ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in increasing amounts of diC4-PI(4,5)P2. The molar ratios of the protein to diC4-PI(4,5)P2 are shown in the inset: 1:0 (black), 1:0.5 (royal blue), 1:1 (turquoise), 1:2 (lime green), 1:4 (orange), 1:6 (pink) and 1:8 (red). ( C ) The chemical shift perturbations (CSPs) of each residue during NMR titrations (up, diC4-PI(3,4,5)P3 titration; down, diC4-PI(4,5)P2 titration) are calculated and shown with the secondary elements on top. White dots indicate pro residues. Black dots indicate residues with no data. The mean value and the mean value plus one standard deviation are indicated by dash and solid lines, respectively. Residues with CSPs between mean value and mean value plus one standard deviation are colored gold, and above mean value plus one standard deviations are colored red. ( D , E ) Surface representations of the Arap3-PH1 structure with the perturbed residues upon binding to diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 are colored and labeled.

**Techniques Used: **Binding Assay, Titration, Standard Deviation, Labeling

# pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis"

**Article Title: **Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis

**Journal: **PLoS ONE

**doi: **10.1371/journal.pone.0055393

**Figure Legend Snippet:**Standard plot of absorbance at 450 nm versus (A) log pmol PI(4,5)P2 and (C) log pmol PI3P. B Percentage change in PI(4,5)P2 content after BL irradiation in control (10 mM PIPES), neomycin (250 µM) and U73122 (25 µM) samples. D Percentage change in PI3P level after BL irradiation in control (10 mM PIPES), WM (10 µM) and LY294002 (200 µM). The vertical bars represent SE of 3 independent experiments.

**Techniques Used: **Irradiation

**Figure Legend Snippet:**Phot1 induction by weak BL activates PI3K and PI4K, thereby producing PI3P and PI4P respectively. PI4P can be hydrolyzed by PLC generating water soluble Ins(1,4)P2. On the other hand, phot2 triggers activation of PI3K, PI4K and the PI(4,5)P2-PLC pathway upon weak BL induction, and only the PI(4,5)P2-PLC pathway upon strong BL. IPPs can be stepwise phosphorylated by inositolpolyphosphate multikinases, IPKs, to produce inositol hexaphosphate (InsP6), which has been also shown to be linked with Ca 2+ mobilization . One of the modes of action for PI3P, PI4P and PI(4,5)P2 during chloroplast relocations is calcium release in the cytoplasm. ↑ represents Ca 2+ transient rise. PIP5K: phosphatidylinositol-4-phosphate 5-kinase and IPK: inositol polyphosphate kinase.

**Techniques Used: **Activation Assay

# pi 4 5 p2 substrate (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2 Substrate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2 substrate/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

# pi 4 5 p 2 dic4 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P 2 Dic4, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p 2 dic4/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "The membrane-actin linker ezrin acts as a sliding anchor"

**Article Title: **The membrane-actin linker ezrin acts as a sliding anchor

**Journal: **Science Advances

**doi: **10.1126/sciadv.abo2779

**Figure Legend Snippet:**Components of T-buffer and S-buffer.

**Techniques Used: **

# pi 4 5 p2 content (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2 Content, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2 content/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

# pi 4 5 p2 content (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2 Content, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2 content/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

# pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

# pi 4 5 p2dic4 p 4504 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2dic4 P 4504, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2dic4 p 4504/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

# pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols"

**Article Title: **T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

**Journal: **The Journal of Biological Chemistry

**doi: **10.1074/jbc.RA118.006524

**Figure Legend Snippet:**T cells generate a different landscape of PIPs in response to TCR signal strength. A, a simplified model of T-cell activation focusing on AKT activation was constructed. Simulations in Matlab were performed where TCR signal strength was modulated by altering the amount of TCR-pMHC in the simulation. B–F, the abundance of PTEN (B), P-PDK1 (C), mTORC2 (C), phosphorylated AKT (D), PI(4,5)P2 (E), and PIP3 (F) were plotted as a function of TCR signal strength. G–I, mass ELISA assays were used to measure the amount of PI(4,5)P2 (G), PIP3 (H), and PI(3,4)P2 (I) generated in murine CD4+ T cells isolated by negative selection stimulated using a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of 10 μm PTEN inhibitor (SF1670). J, CD4+ T cells were nucleofected with either scrambled control (SC) or siRNA targeting PTEN (siRNA) for 48 h, Western blotting was utilized to monitor PTEN levels, and actin was utilized as a loading control. K–M, mass ELISA assays were used to measure the amount of PI(4,5)P2 (K), PIP3 (L), and PI(3,4)P2 (M) generated in murine CD4+ T cells treated with the scrambled control or siRNA targeting PTEN that were activated for 10 min with either a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml). Each experiment was repeated three times, and error bars are ± standard deviation. A two-way ANOVA statistical test was performed. ****, <0.0001; ***, <0.001; **, <0.01; *, <0.05. Symbols over data points are comparisons between the low- and high-dose groups, and symbols in the legend are between the untreated and SF1670-treated groups.

**Techniques Used: **Activation Assay, Construct, Enzyme-linked Immunosorbent Assay, Generated, Isolation, Selection, Western Blot, Standard Deviation

**Figure Legend Snippet:**Weaker TCR signal strengths generate more PI(4,5)P2. Total murine splenocytes were activated with varying doses of plate-bound anti-CD3 antibody indicated in each panel and a constant amount of soluble anti-CD28 antibody (1 μg/ml) for various time points. A, flow cytometry was utilized to measure PI(4,5)P2 in CD4+ T cells stained with an antibody that specifically binds to PI(4,5)P2. CD4+ T cells were defined as cells positive for both TCR and CD4 receptors. B, the percentage of CD4+ T cells positive for PI(4,5)P2 at 10 min of activation were plotted versus anti-CD3 antibody dose. C, the kinetics of PI(4,5)P2 positive CD4+ T-cell formation were plotted for the 0.25 and 1.0 μg/ml anti-CD3 antibody doses. D, the percentage of PI(4,5)P2 positive CD4+ T cells were plotted as a function of both anti-CD3 antibody dose and time. E–G, imaging flow cytometry was utilized to track generation of PI(4,5)P2 (E), PIP3 (F), and PI(3,4)P2 (G) from a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in murine CD4+ T cells isolated by negative selection. A one-way ANOVA was utilized to experiments comparing the impact of α-CD3 antibody dose on PI(4,5)P2 (B). A two-way ANOVA statistical test was performed to analyze the kinetic profiles (C and E–G). For all statistical tests, p values were summarized as follows: ****, <0.0001; ***, <0.001; **, <0.01; *, <0.05. Each experiment was repeated three times, and error bars are ± standard deviation.

**Techniques Used: **Flow Cytometry, Staining, Activation Assay, Imaging, Isolation, Selection, Standard Deviation

**Figure Legend Snippet:**TCR signal strength regulates PIP colocalization to the TCR and stability of TCR capping. The localization of the TCR and either PI(4,5)P2 (A), PIP3 (B), or PI(3,4)P2 (C) was measured in murine CD4+ T cells that were purified by negative selection stimulated using low (0.25 μg/ml), medium (0.5 μg/ml), and high (1.0 μg/ml) doses of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) using imaging flow cytometry. The yellow scale bars correspond to 5 μm. D, the colocalization score between the TCR and PI(4,5)P2, PIP3, and PI(4,5)P2 was calculated in the IDEAS software package from at least 1000 individual cells. E, different levels of TCR capping were observed in the imaging flow cytometry data. The yellow scale bars correspond to 5 μm. F, the Delta centroid function in the IDEAS software package was utilized to calculate the level of TCR capping from the imaging flow cytometry data as a function of activation time in CD4+ T cells that received a weak or strong TCR signal. A one-way ANOVA statistical test was performed to analyze the data in D. A two-way ANOVA statistical test was performed to analyze the kinetic profiles (F). For all statistical tests, p values were summarized as follows: ****, <0.0001; **, <0.01; *, <0.05. Each experiment was repeated three times, and error bars are ± standard deviation.

**Techniques Used: **Purification, Selection, Imaging, Flow Cytometry, Software, Activation Assay, Standard Deviation

**Figure Legend Snippet:**Identification of PIP-binding proteins in naïve CD4+ T cells. A, a proteomics screen was utilized to identify phosphatidylinositol-binding proteins in resting murine CD4+ T cells that were purified by negative selection. B, proteins that bound to beads coated with different PIPs were identified by MS. A label-free approach was utilized to quantitate the relative abundance of each protein bound to PI(4,5)P2, PIP3, or PI(3,4)P2 beads. A protein had to be 3-fold more abundant relative to the other groups to be classified as specifically binding to a particular PIP-coated bead. C, pathway analysis was performed using the lists of proteins that bound to specific PIP beads using the Ingenuity software package. p values were calculated using the right-tailed Fisher's exact test, and the default p value cutoff for significance was <0.05. D–G, the average levels of proteins in each PIP pulldown determined by MS were plotted ± standard deviation from three experiments for proteins that function in signaling (D), chromatin remodeling (E), splicing (F), and transcription (G). A one-way ANOVA statistical test was performed (D–G). For all statistical tests, p values were summarized as follows: ****, <0.0001; ***, <0.001; **, <0.01.

**Techniques Used: **Binding Assay, Purification, Selection, Software, Standard Deviation

**Figure Legend Snippet:**The balance of PI(4,5)P2 versus PIP3 is necessary for interpreting TCR signal strength and setting AKT activation thresholds. A, TCR signaling engages PI3K to generate PIP3, which in turn activates both PDK1 and mTORC2. Pdk1 phosphorylates Ser-308 on AKT, and MTORC2 phosphorylates Thr-473 on AKT, which are required for AKT activation. B, the abundance of PIP3 generated in CD4+ T cells activated for 10 min with varying doses of plate-bound anti-CD3 antibody indicated in each panel and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of 10 μm PTEN inhibitor, SF1670, was determined by a mass ELISA assay from three independent experiments. C, murine CD4+ T cells were activated for 10 min in triplicate with varying doses of plate-bound anti-CD3 antibody and 1 μg/ml anti-CD28 antibody in the presence or absence of 10 μm PTEN inhibitor (SF1670). D, the abundance of PTEN as a function of anti-CD3 antibody concentration was determined by densitometry. E and F, P-PDK1, p-RICTOR, p-AKT308, and p-AKT473 Western blots were quantitated by densitometry where all phosphorylated species were normalized to the total amount of the respective protein. G and H, the normalized abundance of p-PDK1, p-RICTOR, p-AKT308, and p-AKT473 determined by Western blotting was plotted versus the level of PIP3 generated.

**Techniques Used: **Activation Assay, Generated, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot

**Figure Legend Snippet:**Model describing how the balance of PI(4,5)P2/PIP3 is used to measure TCR signal strength. Stimulation of a T cell with a weak TCR signal results in maintenance of PTEN, elevated PI(4,5)P2, and lower PIP3 levels. In this mode, there is sufficient PIP3 generation to activate PDK1 to phosphorylate AKT on Thr-308. The elevated PI(4,5)P2 levels generated from a weak signal activate FAK. Stimulation with a strong TCR signal reduces PTEN levels, which allows for higher PIP3 levels and reduced PI(4,5)P2. Higher levels of PIP3 activate both PDK1 and mTORC2, which results in phosphorylation of AKT on both Thr-308 and Ser-473. Diminished PI(4,5)P2 results in weak FAK activation. The AKT proteoforms generated by a weak versus high TCR signal have different substrate specificities and activate divergent downstream signaling pathways to program different T-cell fate decisions.

**Techniques Used: **Generated, Activation Assay

# pi 4 5 p2 (Echelon Biosciences)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

## Structured Review

Pi 4 5 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/pi 4 5 p2/product/Echelon Biosciences

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols"

**Article Title: **T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

**Journal: **The Journal of Biological Chemistry

**doi: **10.1074/jbc.RA118.006524

**Figure Legend Snippet:**T cells generate a different landscape of PIPs in response to TCR signal strength. A, a simplified model of T-cell activation focusing on AKT activation was constructed. Simulations in Matlab were performed where TCR signal strength was modulated by altering the amount of TCR-pMHC in the simulation. B–F, the abundance of PTEN (B), P-PDK1 (C), mTORC2 (C), phosphorylated AKT (D), PI(4,5)P2 (E), and PIP3 (F) were plotted as a function of TCR signal strength. G–I, mass ELISA assays were used to measure the amount of PI(4,5)P2 (G), PIP3 (H), and PI(3,4)P2 (I) generated in murine CD4+ T cells isolated by negative selection stimulated using a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of 10 μm PTEN inhibitor (SF1670). J, CD4+ T cells were nucleofected with either scrambled control (SC) or siRNA targeting PTEN (siRNA) for 48 h, Western blotting was utilized to monitor PTEN levels, and actin was utilized as a loading control. K–M, mass ELISA assays were used to measure the amount of PI(4,5)P2 (K), PIP3 (L), and PI(3,4)P2 (M) generated in murine CD4+ T cells treated with the scrambled control or siRNA targeting PTEN that were activated for 10 min with either a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml). Each experiment was repeated three times, and error bars are ± standard deviation. A two-way ANOVA statistical test was performed. ****, <0.0001; ***, <0.001; **, <0.01; *, <0.05. Symbols over data points are comparisons between the low- and high-dose groups, and symbols in the legend are between the untreated and SF1670-treated groups.

**Techniques Used: **Activation Assay, Construct, Enzyme-linked Immunosorbent Assay, Generated, Isolation, Selection, Western Blot, Standard Deviation

**Figure Legend Snippet:**Weaker TCR signal strengths generate more PI(4,5)P2. Total murine splenocytes were activated with varying doses of plate-bound anti-CD3 antibody indicated in each panel and a constant amount of soluble anti-CD28 antibody (1 μg/ml) for various time points. A, flow cytometry was utilized to measure PI(4,5)P2 in CD4+ T cells stained with an antibody that specifically binds to PI(4,5)P2. CD4+ T cells were defined as cells positive for both TCR and CD4 receptors. B, the percentage of CD4+ T cells positive for PI(4,5)P2 at 10 min of activation were plotted versus anti-CD3 antibody dose. C, the kinetics of PI(4,5)P2 positive CD4+ T-cell formation were plotted for the 0.25 and 1.0 μg/ml anti-CD3 antibody doses. D, the percentage of PI(4,5)P2 positive CD4+ T cells were plotted as a function of both anti-CD3 antibody dose and time. E–G, imaging flow cytometry was utilized to track generation of PI(4,5)P2 (E), PIP3 (F), and PI(3,4)P2 (G) from a low (0.25 μg/ml) or high (1.0 μg/ml) dose of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in murine CD4+ T cells isolated by negative selection. A one-way ANOVA was utilized to experiments comparing the impact of α-CD3 antibody dose on PI(4,5)P2 (B). A two-way ANOVA statistical test was performed to analyze the kinetic profiles (C and E–G). For all statistical tests, p values were summarized as follows: ****, <0.0001; ***, <0.001; **, <0.01; *, <0.05. Each experiment was repeated three times, and error bars are ± standard deviation.

**Techniques Used: **Flow Cytometry, Staining, Activation Assay, Imaging, Isolation, Selection, Standard Deviation

**Figure Legend Snippet:**TCR signal strength regulates PIP colocalization to the TCR and stability of TCR capping. The localization of the TCR and either PI(4,5)P2 (A), PIP3 (B), or PI(3,4)P2 (C) was measured in murine CD4+ T cells that were purified by negative selection stimulated using low (0.25 μg/ml), medium (0.5 μg/ml), and high (1.0 μg/ml) doses of plate-bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 μg/ml) using imaging flow cytometry. The yellow scale bars correspond to 5 μm. D, the colocalization score between the TCR and PI(4,5)P2, PIP3, and PI(4,5)P2 was calculated in the IDEAS software package from at least 1000 individual cells. E, different levels of TCR capping were observed in the imaging flow cytometry data. The yellow scale bars correspond to 5 μm. F, the Delta centroid function in the IDEAS software package was utilized to calculate the level of TCR capping from the imaging flow cytometry data as a function of activation time in CD4+ T cells that received a weak or strong TCR signal. A one-way ANOVA statistical test was performed to analyze the data in D. A two-way ANOVA statistical test was performed to analyze the kinetic profiles (F). For all statistical tests, p values were summarized as follows: ****, <0.0001; **, <0.01; *, <0.05. Each experiment was repeated three times, and error bars are ± standard deviation.

**Techniques Used: **Purification, Selection, Imaging, Flow Cytometry, Software, Activation Assay, Standard Deviation

**Figure Legend Snippet:**Identification of PIP-binding proteins in naïve CD4+ T cells. A, a proteomics screen was utilized to identify phosphatidylinositol-binding proteins in resting murine CD4+ T cells that were purified by negative selection. B, proteins that bound to beads coated with different PIPs were identified by MS. A label-free approach was utilized to quantitate the relative abundance of each protein bound to PI(4,5)P2, PIP3, or PI(3,4)P2 beads. A protein had to be 3-fold more abundant relative to the other groups to be classified as specifically binding to a particular PIP-coated bead. C, pathway analysis was performed using the lists of proteins that bound to specific PIP beads using the Ingenuity software package. p values were calculated using the right-tailed Fisher's exact test, and the default p value cutoff for significance was <0.05. D–G, the average levels of proteins in each PIP pulldown determined by MS were plotted ± standard deviation from three experiments for proteins that function in signaling (D), chromatin remodeling (E), splicing (F), and transcription (G). A one-way ANOVA statistical test was performed (D–G). For all statistical tests, p values were summarized as follows: ****, <0.0001; ***, <0.001; **, <0.01.

**Techniques Used: **Binding Assay, Purification, Selection, Software, Standard Deviation

**Figure Legend Snippet:**The balance of PI(4,5)P2 versus PIP3 is necessary for interpreting TCR signal strength and setting AKT activation thresholds. A, TCR signaling engages PI3K to generate PIP3, which in turn activates both PDK1 and mTORC2. Pdk1 phosphorylates Ser-308 on AKT, and MTORC2 phosphorylates Thr-473 on AKT, which are required for AKT activation. B, the abundance of PIP3 generated in CD4+ T cells activated for 10 min with varying doses of plate-bound anti-CD3 antibody indicated in each panel and a constant amount of soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of 10 μm PTEN inhibitor, SF1670, was determined by a mass ELISA assay from three independent experiments. C, murine CD4+ T cells were activated for 10 min in triplicate with varying doses of plate-bound anti-CD3 antibody and 1 μg/ml anti-CD28 antibody in the presence or absence of 10 μm PTEN inhibitor (SF1670). D, the abundance of PTEN as a function of anti-CD3 antibody concentration was determined by densitometry. E and F, P-PDK1, p-RICTOR, p-AKT308, and p-AKT473 Western blots were quantitated by densitometry where all phosphorylated species were normalized to the total amount of the respective protein. G and H, the normalized abundance of p-PDK1, p-RICTOR, p-AKT308, and p-AKT473 determined by Western blotting was plotted versus the level of PIP3 generated.

**Techniques Used: **Activation Assay, Generated, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot

**Figure Legend Snippet:**Model describing how the balance of PI(4,5)P2/PIP3 is used to measure TCR signal strength. Stimulation of a T cell with a weak TCR signal results in maintenance of PTEN, elevated PI(4,5)P2, and lower PIP3 levels. In this mode, there is sufficient PIP3 generation to activate PDK1 to phosphorylate AKT on Thr-308. The elevated PI(4,5)P2 levels generated from a weak signal activate FAK. Stimulation with a strong TCR signal reduces PTEN levels, which allows for higher PIP3 levels and reduced PI(4,5)P2. Higher levels of PIP3 activate both PDK1 and mTORC2, which results in phosphorylation of AKT on both Thr-308 and Ser-473. Diminished PI(4,5)P2 results in weak FAK activation. The AKT proteoforms generated by a weak versus high TCR signal have different substrate specificities and activate divergent downstream signaling pathways to program different T-cell fate decisions.

**Techniques Used: **Generated, Activation Assay